Single-cell embryonic reporter assays to study tissue-specificity - PROJECT SUMMARY In response to the FOA: Focused Technology research and development, PAR-25-203, we aim to develop episomal single-cell reporter assays that will allow researchers the unprecedented ability to measure the activity of 100-1000s of regulatory elements with tissue-specific resolution in whole developing embryos. While our technology can be used to study a variety of regulatory elements (enhancers, promoters, silencers, and combinations of these elements) our initial application of this technology will focus on enhancers. Enhancers are a critical regulatory element controlling the timing and location of gene expression. Most mutations associated with disease, evolutionary adaptations, and phenotypic diversity reside in enhancers, yet we have difficulty determining causal variants and their location of action. Enhancers are also used as tissue and temporal-specific drivers to generate conditional transgenic mice, yet current drivers are crude, and greater precision is required to interrogate genetic function. There are millions of untested putative enhancers, and we are quickly amassing more predictions of putative enhancers and disease variants. Given the centrality of enhancers to successful development, disease and biomedical research, it is critical we develop high-throughput methods to interrogate the spatiotemporal activity of enhancers at scale. To date, Single-cell Massively Parallel Reporter Assays (MPRAs) to study regulatory elements have only been implemented within the context of tissue culture, embryoid bodies, and limited tissues. Previously, the lack of whole organism studies and focus on limited number of cells have led to misconceptions regarding enhancers and how tissue-specificity is encoded within genomes. Current technologies do not provide whole organism tissue-specific read-outs of enhancer activity at scale. This technological gap is stalling our ability to interpret genomic data, test predictions regarding enhancers and enhancer variants, understand development, and engineer tissue-specific enhancers for precise conditional transgenic mice. To date, high-throughput whole embryo MPRAs have been limited to Ciona robusta (Ciona), which we developed. Drawing on our expertise developing MPRAs in Ciona and chick, and expertise in mouse development, we propose the development of single-cell embryonic MPRAs (SEMPRAs), with both tissue- specific and single cell resolution in developing Ciona (Aim 1), chick (Aim 2) and mouse (Aim 3) embryos. These aims depend on efficient delivery of SEMPRA libraries into all cells of the developing embryo, sufficient plasmids being present in cells as the embryos develop, and methods to carefully measure all tested elements. We have innovative solutions to achieve these goals that rely on our published work, preliminary data, and novel use of established technologies. This proposal will use validated enhancers to optimize the SEMPRA technology. Successful completion of this proposal will result in prototype-ready versions of these SEMPRA technologies that can analyze 100s-1000s of elements in whole Ciona, chick and mouse embryos.
